Focal adhesion kinase (FAK) is important to cellular functions such as proliferation, migration, and survival of anchorage-dependent cells. We investigated the role of FAK in modulating normal cellular responses, specifically cell survival in response to inflammatory stimuli and serum withdrawal, using FAK-knockout (FAK(-/-)) embryonic fibroblasts. FAK(-/-) fibroblasts were more vulnerable to TNF-alpha-induced apoptosis, as measured by terminal deoxynucleotidyl transferase positivity. FAK(-/-) fibroblasts also demonstrated increased procaspase-3 cleavage to p17 subunit, whereas this was undetectable in FAK(+/+) fibroblasts. Insulin receptor substrate-1 expression was completely abolished and NF-kappaB activity was reduced, with a concomitant decrease in abundance of the anti-apoptotic protein Bcl-x(L) in FAK(-/-) cells. Upon serum withdrawal, FAK(+/+) cells exhibited marked attenuation of basal ERK phosphorylation, while FAK(-/-) cells, in contrast, maintained high basal ERK phosphorylation. Moreover, inhibition of ERK phosphorylation potentiated serum withdrawal-induced caspase-3 activity. This was paralleled by increased insulin receptor substrate (IRS)-2 expression in FAK(-/-) cells, although both insulin- and IGF-1-mediated phosphorylation of Akt/PKB and GSK-3 were impaired. This suggests that IRS-2 protects against apoptosis upon serum withdrawal via the ERK signaling pathway. The specific role of FAK to protect cells from apoptosis is regulated by activation and phosphorylation of NF-kappaB and interaction between activated growth factor anti-apoptotic signaling pathways involving both phosphatidylinositol 3-kinase/Akt and MAPK/ERK1/2. We demonstrate that FAK is necessary for upregulation of the anti-apoptotic NF-kappaB response, as well as for normal expression of growth factor signaling proteins. Thus we propose a novel role for FAK in protection from cytokine-mediated apoptosis.

Integrins mediate interactions between cells and extracellular matrix proteins that modulate growth factor signaling. Focal adhesion kinase (FAK) is a key multifunctional integrin pathway protein. We recently reported that disruption of FAK impairs insulin-mediated glycogen synthesis in hepatocytes. To test the hypothesis that FAK regulates skeletal muscle insulin action, we reduced FAK expression in L6 myotubes using FAK antisense. In untransfected myotubes, insulin stimulated both FAK tyrosine phosphorylation and kinase activity. Cells treated with antisense FAK showed 78 and 53% reductions in FAK mRNA and FAK protein, respectively, whereas insulin receptor substrate 1/2 and paxillin abundance were unaffected. Insulin-stimulated U-(14)C-glucose incorporation into glycogen was abolished by FAK antisense, and 2-deoxy-glucose uptake and glucose transporter 4 (GLUT4) translocation were both markedly attenuated. Antisense FAK did not alter GLUT1 or GLUT3 protein abundance. Immunofluorescence staining showed decreased FAK Tyr(397) phosphorylation and reduced actin stress fibers. Thus, in skeletal myotubes, FAK regulates the insulin-mediated cytoskeletal rearrangement essential for normal glucose transport and glycogen synthesis. Integrin signaling may play an important regulatory role in muscle insulin action.

Obesity is central to the development of the insulin resistance metabolic syndrome, 1 which is characterized by hyperinsulinaemia, hyperglycaemia, hypertension, dyslipidaemia and vascular endothelial dysfunction, which predispose to atherosclerosis. [2][3][4][5] The fatty Zucker rat constitutes an animal model of the metabolic syndrome, since it becomes obese and develops insulin resistance, hyperinsulinaemia and hypertension. It is therefore useful for the study of abnormalities of vascular reactivity seen in obesity and insulin resistance. [6][7][8] TZDs, which are antidiabetic, insulin-sensitizing drugs, have been shown to ameliorate hyperglycaemia in rodent models of insulin resistance and diabetes mellitus as well as in humans.9-17 TZDs are high-affinity ligands for PPARγ, 18 and have also been shown to have vascular protective effects, preventing the development of atherosclerosis and neointimal thickening, 19 and have recently been shown to improve endothelial function.20-23 PPARγ ligands exhibit anti-inflammatory effects, including attenuation of cytokine production e.g. tumour necrosis factor-α and interleukin-6; and inhibition of VCAM-1 and ICAM-1. 1,21,24,25 In coronary arteries, TZDs reduce oxidative stress, improving the NO-vasoconstrictor balance. 26 We therefore tested the hypothesis that the antihyperglycaemic and antihypertensive actions of pioglitazone are associated with improved vascular reactivity. We find that pioglitazone ameliorated metabolic insulin resistance, protected against elevated BP and markedly improved conductance artery SOD-mediated vasorelaxation. To test the hypothesis that the thiazolidinedione agent, pioglitazone, mediates its chronic BP lowering action via improving vascular reactivity. Methods and Results: Lean (Fa/fa) and obese (fa/fa) Zucker rats were treated with or without pioglitazone (20 mg/ kg/day) for 4 weeks (n=8 animals per group). Pioglitazone treatment was associated with a significant improvement in oral glucose tolerance in the obese animals (p<0.05 compared with untreated obese). Pioglitazone prevented the development of hypertension seen in obese untreated rats (SBP 126±1 versus 138±1 mmHg; p<0.0001). Aortic ring preparations from pioglitazone-treated obese rats showed improved relaxation responsiveness (ED 50 0.28 versus 1.15 U/ ml, p<0.001) to SOD, a NO potentiator, compared with untreated obese animals. Conclusions: SOD-mediated vasorelaxation may contribute to the chronic antihypertensive effect and/or the improvement in insulin sensitivity following pioglitazone treatment.